An elevation of cytosolic free calcium concentration is an integral component of the mechanism by which cells respond to hormones, growth factors and neurotransmitters. D-myo-inositol 1,4,5-trisphosphate ( IP3 ) is an intracellular messenger mediating the mobilization of Ca2+ from intracellular stores by interaction with an ubiquitous receptor ( IP3R ) that acts as a ligand-gated Ca2+ channel. IP3Rs are redox sensitive channels and are sensitized by oxidative stress. However, the molecular basis of this regulation is poorly understood. Ca2+ released from IP3Rs is locally transmitted to the mitochondria and can stimulate metabolism, and in higher amounts, can also initiate cell death. The overarching hypothesis of this study is that redox modulation of IP3Rs is an important component of the regulation of Ca2+ signals in cell death pathways. The proposal encompasses the following three specific aims: 1] To measure and map redox changes in IP3Rs. We have developed methods to determine the redox state of IP3Rs in vivo which will be used to quantitate the effects of exogenous and endogenous agents causing oxidative stress. Preliminary studies using mass-spectroscopy identify a subset of 11 cysteines that become oxidized in IP3R-1. The type of oxidative modifications occurring will be identified. Redox-sensitive thiols will be mutate and the functional sensitivity to oxidative stress will be assessed. 2] To measure IP3R redox state at the ER/mitochondrial junction. We will test the hypothesis that the pool of IP3Rs located at the ER/mito junction is particularly prone to ROS modifications. We will employ subcellular fractionation and imaging methods utilizing targeted IP3Rs, ROS-sensitive fluorescent proteins, ROS-producing photosensitive probes and targeted catalases. 3] To investigate the role of IP3R redox changes in models of ER stress/apoptosis. We will test the hypothesis that ER-resident NADPH oxidases play an important role in IP3R redox regulation. Liver will be used as an experimental model to induce ER stress. The role of IP3R redox regulation in ER stress pathways activated by fructose will be examined. The long-term goal of the proposal is to obtain a detailed understanding of how oxidative stress impacts intracellular Ca2+ signaling under normal and disease conditions.